Project Summary/Abstract The objective of this proposal is to elucidate how glucose metabolism (i.e. glycolysis and gluconeogenesis) and mitochondrial metabolism are spatially and functionally (spatiofunctionally) interconnected and dynamically orchestrated in human living cells. Current understanding of the highly enmeshed web of metabolic pathways has been limited in 2D, but cellular metabolism takes place in space and time (i.e. 4D). A number of metabolic pathways are spatially confined into either membrane-bound organelles or membraneless compartments. To understand how metabolic pathways are regulated and networked, it is vital to know how organelles or membraneless compartments are spatially arranged and functionally interplay in 4D. We have recently reported that the cytoplasmic, rate-determining enzymes in glucose metabolism are spatially organized into membraneless compartments in various sizes in human cells. We proposed that they shunt glucose flux to anabolic biosynthetic pathways. More importantly, our preliminary results suggest now that the enzyme compartments, formed by liquid-liquid phase separation (LLPS), might be spatially and functionally linked with mitochondria. Thus, in this proposal, we hypothesize that the enzyme compartments in glucose metabolism are spatially and functionally associated with mitochondria in 4D, by which LLSP plays a role to adapt cellular demands. .We will characterize precise mechanisms of the formation and modulation of the enzyme compartments. In addition, we will reveal how the enzyme compartments of glucose metabolism are functionally and spatially coordinated with the mitochondria and the regulatory mechanisms of their network in living cells. The proposed work will offer new, powerful 4D imaging and analysis approaches to explore novel perspectives of spatiotemporal dynamics of metabolic networks inside cells. This work will provide the fundamental principle of understanding the new class of essential organelles in the cell and will provide a new paradigm to comprehend 4-D map of metabolic networks in living cells.